The present invention relates to improved methods for the identification and quantification of soluble analytes in a sample. More particularly, the present invention relates to a simple rapid flow cytometric method of such analysis.
Simple methods for the analysis and quantification of soluble analytes in a sample, for which analytes specifically binding ligands exist, are useful in a wide variety of applications. Medical applications such as diagnosis of diseases characterized by the presence of certain analytes in a biological sample or the evaluation of commercial or industrial samples containing analytes can be accomplished with such methods. For example, methods for the analysis of soluble analytes, such as antigenic receptors which are shed from their normal positions on the surfaces of formed bodies, particularly cells, can provide information essential to the diagnosis of disease states and the monitoring of the efficacy of treatment of various diseases.
Conventional methods for such analysis included steps such as equilibration of a labeled ligand with a cell suspension, followed by a separation (e.g., wash) of bound from unbound ligands to obtain the solution concentration of ligand. Thereafter graphical analysis employing a set of cell-bound, labeled ligands, fluorescence intensities and the solution concentration of labeled ligand, produced the necessary data for analysis. Other known methods for the analysis of receptors employed differently labeled ligands, external standards for calibration, or image analysis.
Flow cytometry methods were recently used to analyze quantitatively for shed antigenic receptor in solution in a competitive binding experiment that used both unlabeled and labeled ligand with unknown amounts of bound and shed antigen without a separation step or an external standard [Siiman, O. and Burshteyn, A., Cytometry, 40: 316-326 (2000); and U.S. Pat. No. 5,814,468, both incorporated herein by reference]. Briefly described, this method obtained the amount of soluble antigenic receptor in a sample that contained unknown amounts of soluble (i.e., shed) antigenic receptor and cell-surface bound antigenic receptor. The method combined the data from a number of assay steps. In the first assay step, a control sample containing only labeled ligand was mixed and equilibrated with a fixed number of cells in whole blood. In a second step, a competition test sample containing both labeled and unlabeled ligand were mixed sequentially and equilibrated with a fixed number of cells in whole blood. In both steps, the cells were fixed by lysing the red blood cells and quenching (Q-PREPing) them. The cells were run on a conventional flow cytometer. The results were analyzed by determining the unlabeled ligand concentration at which the fluorescence intensity of the labeled ligand in the competitive binding assay using variable titers of unlabeled ligand and fixed titers of labeled ligand matches the fluorescence intensity of the control with no unlabeled ligand. This method permitted an estimation of the concentration of soluble antigenic receptor in blood plasma.
Additional or modified methods for analysis of such bound and shed antigenic receptors are needed for other purposes, such as the measurement of certain antigenic receptors on neutrophils, which have some unusual functions [Edwards S W, xe2x80x9cBiochemistry and physiology of the neutrophilxe2x80x9d, Cambridge, UK: Cambridge, University Press; (1994); Abramson J S, and Wheeler J G, eds, xe2x80x9cThe neutrophil.xe2x80x9d Oxford, UK: IRL Press at Oxford University Press (1993)]. For example, the quantification of the shedding of the Fcxcex3 receptor III on neutrophils in whole blood has thus far been done using a long and tedious ELISA method. Controls are not easily obtained for the ELISA assay.
For example, the Fcxcex3 receptor IIIB is anchored to the neutrophil membrane via a glycosyl-phosphatidylinositol (GPI) linkage through a terminal serine residue. This linkage can be easily cleaved by enzymes such as elastase and pronase or activation by certain stimuli such as the chemotactic peptide, N-formyl-MET-LEU-PHE, and phorbol myristate acetate (PMA), which induce shedding of Fcxcex3 RIIIB from the neutrophil surface [Huizinga T. W. J. et al, J. Clin. Invest., 86: 416-423 (1990); Tosi, M. F. and Zakem, H., J. Clin. Invest., 90: 462-470 (1992)]. Soluble Fcxcex3 RIIIB in normal human serum was detected in widely variable concentrations ranging from 7.3 to 75.9 nmol/L [Fleit H. B., et al, Blood, 79:2721-2728 (1992)] with the monoclonal antibodies, CLBFcRgran 1, BW209/2, and 3G8 or 3G8 Fab.
Thus, there remains a need in the art for additional and modified, flow cytometric methods of analysis for soluble analyte in a sample.
In one aspect, the present invention provides a method of analyzing the concentration or amount of soluble analyte in a sample. The method involves the steps of adding to a series of test samples containing an unknown concentration of soluble analyte: a predetermined amount of formed bodies to which are attached at least one analyte, varying known concentrations of an unlabeled ligand that binds to said analyte, and a known concentration of said ligand labeled with a detectable marker. Preferably, the analytes are covalently attached to the formed bodies and a constant known concentration of the ligand labeled with a detectable marker is used. The samples are incubated and labeled ligand and the unlabeled ligand compete for binding to the formed body-bound analyte and the soluble analyte. The control samples contain the sample with an unknown concentration of soluble analyte, the predetermined amount of the formed bodies, and varying known concentrations of the labeled ligand with no unlabeled ligand. All control samples are incubated to achieve equilibrium binding of ligand to analyte from about 1% occupation of binding sites on analytes bound to the formed bodies to 100% occupation at saturation, i.e., labeled ligand binds to the formed body-bound analytes and the soluble analyte. Each test sample and control sample is analyzed by an instrument that measures detectable signal produced from the labeled ligands bound to the bound analytes on the formed bodies. Thereafter, the intersection of a curve formed by plotting the signal against the concentration of the labeled ligand in the control samples, and a second curve formed by plotting the signal against the concentration of total labeled and unlabeled ligand in the test samples is identified. Preferably, the curve is a linearxe2x80x94log graphical dependence curve and the concentration is a molar concentration. The concentration of unlabeled ligand that bound to soluble analytes in the test samples is determined by evaluating the difference D between the molar concentration that corresponds with the intersection and the constant labeled ligand concentration in the test samples. The concentration of the soluble analytes in the sample is obtained by determining the product of the value D and the binding stoichiometry of the ligand to soluble analytes.
In another aspect, the present invention provides a method of analyzing the concentration or amount of soluble receptors in a sample, wherein the sample comprises formed bodies having the receptors bound thereto and the soluble receptors shed from the formed bodies. The method involves incubating a series of test samples containing a predetermined amount of the formed bodies, varying known concentrations of a ligand that binds to the receptor, and a known concentration of the ligand labeled with a detectable marker. Preferably, a constant known concentration of the ligand labeled with a detectable marker is used. The labeled ligand and the unlabeled ligand compete for binding to the bound and soluble receptors. Control samples contain the sample with an unknown concentration of soluble analyte, the predetermined amount of the formed bodies, and varying known concentrations of the labeled ligand with no unlabeled ligand. All control samples are incubated to achieve equilibrium binding from about 1% occupation of binding sites on formed bodies to 100% occupation at saturation, i.e., labeled ligand binds to the formed body-bound analytes and the soluble analyte. Each test sample and control sample is analyzed in an instrument that measures detectable signal produced from the labeled ligands bound to the bound receptors on the formed bodies. The intersection of a curve formed by plotting the signal against the concentration of the labeled ligand in the control samples, and a second curve formed by plotting the signal against the concentration of total labeled and unlabeled ligand in the test samples is identified. Preferably, the curve is a linearxe2x80x94log graphical dependence curve and the concentration is a molar concentration. The concentration of soluble receptors released from the formed bodies is thereafter determined by evaluating the difference between the concentration that corresponds with the intersection and the constant labeled ligand concentration in the test samples with the binding stoichiometry of the ligand.
In still another aspect, the invention provides a method of diagnosing a disease characterized by an altered level of soluble analyte a biological sample, e.g., blood, serum, tissue, of a mammal. The method involves analyzing the amount of soluble analyte by performing the steps of the above-described method, and comparing the concentration of soluble analyte from the mammal""s samples with known normal concentrations of soluble analyte in samples from a healthy mammal, wherein a difference between the concentration of the soluble analyte in the test mammal""s sample and the normal concentrations is indicative of disease.
In yet another aspect, the invention provides a method for diagnosing a disease characterized by an altered level of receptors shed by target cells into the blood of a mammal comprising the steps of analyzing the amount of soluble receptor shed from the surface of the target cells into the mammalian blood sample by the method described above; and comparing the concentration of soluble receptors from the mammal""s samples with known normal concentrations of soluble receptors in samples of healthy mammalian blood. A significant difference between the concentration of soluble receptors in the mammal""s blood sample and the normal concentrations is indicative of disease.
In another aspect, the invention provides a computer program which identifies and analyzes the amount of soluble analyte in a sample, e.g., soluble receptor shed from the surface of a formed body into solution in a sample comprising formed bodies, by implementing the above-mentioned method.
In still another aspect, the invention provides an analysis instrument that comprises an integrated computer program which identifies and analyzes the amount of soluble analyte in a sample, e.g., soluble receptor shed from the surface of a formed body into solution in a sample comprising formed bodies, by implementing the above-mentioned method.
In another aspect, the invention provides a diagnostic kit useful for the performance of these methods, particularly for diagnosis of a disease.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.